Biosynthesis of enterobacterial common antigen in Escherichia coli. Biochemical characterization of Tn10 insertion mutants defective in enterobacterial common antigen synthesis.

Abstract

Twelve independent Tn10 insertion mutants of Escherichia coli K12 were isolated that were defective in the synthesis of enterobacterial common antigen (ECA). The mutants were identified by screening a random pool of Tn10 insertion mutants for their ECA phenotype using a colony-immunoblot assay. All 12 of the Tn10 insertion mutants were found to be located in the chromosomal region of the rff-rfe genes. Four of the Tn10 insertions were in rfe genes while the remaining eight Tn10 insertions were in rff genes. All of the rfe::Tn10 insertion mutants were defective in the synthesis of GlcNAc-pyrophosphorylundecaprenol (C55-PP-GlcNAc, lipid I), the first lipid-linked intermediate involved in ECA synthesis. Biochemical characterization of the rff::Tn10 insertion mutants revealed that they were defective in various steps of ECA synthesis subsequent to the synthesis of lipid I. These defects included: (i) the inability to synthesize UDP-ManNAcA due to Tn10 insertions in the structural genes for UDP-GlcNAc-2-epimerase (rffE) and UDP-ManNAcA (N-acetyl-D-mannosaminuronic acid) dehydrogenase (rffD), (ii) defects in the synthesis of C55-GlcNAc-ManNAcA (lipid II) due to insertion of transposon Tn10 in the structural gene for the UDP-ManNAcA transferase (rffM), (iii) the inability to synthesize TDP-Fuc4NAc (4-acetamido-4,6-dideoxy-D-galactose) due to Tn10 insertions in the structural gene for the transaminase that catalyzes the conversion of TDP-4-keto-6-deoxy-D-glucose to TDP-4-amino-4,6-dideoxy-D-galactose (rffA), and (iv) defects in steps subsequent to the synthesis of C55-GlcNAc-ManNAcA-Fuc4NAc (lipid III). In addition, a re-examination of a mutant possessing the rff-726 lesion revealed that it was defective in the synthesis of lipid III due to a defect in the structural gene for the Fuc4NAc transferase (rffT).